Tau is a microtubule-associated protein that is abnormally phosphorylated and aggregates in Alzheimer?s disease (AD) and AD-related dementias, leading to its impaired functionality. No disease modifying treatment exists for these diseases, making them a significant healthcare priority. Though we have known for decades that several modifications are intimately associated with sporadic disease and mutations in tau directly cause inherited degenerative tauopathies, the precise molecular pathways engaged by pathological tau proteins to actively cause neurodegeneration are unknown. This knowledge gap remains a significant and critical problem that this proposal aims to address. Current thinking in the field suggests that disease-related tau modifications exert toxicity by disruption of microtubules and/or by enhancing tau aggregation. However, evidence from our group and others supports an alternative explanation. That is, tau acts to regulate signaling pathways and tau toxicity is due to an aberration of this function. Using the isolated axoplasm from squid giant axons, we discovered a functional signaling motif in the N-terminus of tau called the phosphatase-activating domain (PAD) that activates a protein phosphatase-1 (PP1)-dependent signaling cascade. We also identified that known pathological changes in tau (e.g. phosphorylation and oligomerization) alter tau?s structure leading to aberrant PAD-dependent activation of this pathway and axonal toxicity. We propose to focus on determining whether tau normally regulates PP1-dependent functions in neurons and on PP1-dependent pathways of tau toxicity. Our central hypothesis is that, through a PP1-dependent mechanism, tau normally regulates neuronal function and health, but in disease tau causes axonal, synaptic and/or neuronal toxicity through aberrations of this mechanism. In Aim 1, we will test the hypothesis that specific motifs within tau and PP1 are critical for the interaction with and activation of PP1 by tau proteins. We will use a combination of point mutations/deletions to map the specific domains in tau and PP1 isoforms that underlie tau?s ability to bind and activate PP1. In Aim 2, we will test the hypothesis that tau localizes with PP1 in specific subcellular compartments where it regulates PP1-dependent pathways. Here, we will use novel human tau-KI mouse primary neuron cultures and a combination of super-resolution microscopy, subcellular fraction and other protein-protein interaction assays to determine where in neurons tau and PP1 interact. Also, we will knockdown tau to determine its functional relationship to multiple PP1-dependent pathways in neurons. In Aim 3, we will test the hypothesis that disease forms of tau drive toxicity via PP1-dependent mechanisms. We will use a novel tau pre-formed fibril seeding model in the hTau-KI mice as well as the PS19 mutant tau mouse line to provide the critical in vivo translational insights for the tau-PP1 relationship and how disease forms of tau lead to PP1-dependent toxicity. Together, the proposed studies will fill critical gaps in our knowledge and will produce a significant impact by identifying important physiological and pathological roles for tau in regulating PP1 in neurons.